The unusual tetrasaccharide sequence GlcAβ-3GalNAc(4-sulfate)(β1–4GlcA(2-sulfate)β1–3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D
Eight hexasaccharide fractions were isolated from commercial shark cartilage chondroitin sulfate D by means of gel nitration chromatography and HPLC on an amine-bound silica column after exhaustive digestion with sheep testicular hyaluronidase. Capillary electrophoresis of the enzymatic digests as well as one- and two-dimensional 500 MHz 1H-NMR spectroscopy demonstrated that these hexasaccharides share the common core saccharide structure GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc with three, four, or five sulfate groups in different combinations. Six structures had the same sulfation profiles as those of the unsaturated hexasaccharides isolated from the same source after digestion with chondroitinase ABC (Sugahara et al., Eur. J. Biochem., 293, 871–880, 1996) and the other two have not been reported so far. In the new components, a D disaccharide unit, GlcA(2-sulfate)β1-3GalNAc(6-sulfate), characteristic of chondroitin sulfate D was arranged on the reducing side of an A disaccharide unit, GlcAβ1-3GalNAc(4-sulfate), forming an unusual A-D tetrasaccharide sequence, GlcAβ1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)β1-3GaINAc(6-sulfate) which is known to be recognized by the monoclonal antibody MO225. These findings support the notion that the tetrasaccharide sequence, GlcAβ1-3GalNAc(4-sulfate)β1-4GlcAβ1-3GalNAc(6-sulfate) is included in the acceptor site of a hitherto unreported 2-O-sulfotransferase responsible for its synthesis. The sulfated hexasaccharides isolated in this study will be useful as authentic oligosaccharide probes and enzyme substrates in studies of sulfated glycosaminogly-cans.